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Nanolipoprotein particles (NLPs), also called “nanodiscs”, are discoidal particles with a patch of lipid bilayer corralled by apolipoproteins. NLPs have long been of interest due to both their utility as membrane-model systems into which membrane proteins can be inserted and solubilized and their physiological role in lipid and cholesterol transport via high-density lipoprotein (HDL) and low-density lipoprotein (LDL) maturation, which are important for human health. Serial femtosecond crystallography (SFX) at X-ray free electron lasers (XFELs) is a powerful approach for structural biology of membrane proteins, which are traditionally difficult to crystallize as large single crystals capable of producing high-quality diffraction suitable for structure determination. To facilitate understanding of the specific role of two apolipoprotein/lipid complexes, ApoA1 and ApoE4, in lipid binding and HDL/LDL particle maturation dynamics, and to develop new SFX methods involving NLP membrane protein encapsulation, we have prepared and crystallized homogeneous populations of ApoA1 and ApoE4 NLPs. Crystallization of empty NLPs yields semi-ordered objects that appear crystalline and give highly anisotropic and diffuse X-ray diffraction, similar to fiber diffraction. Several unit cell parameters were approximately determined for both NLPs from these measurements. Thus, low-background, sample conservative methods of delivery are critical. Here we implemented a fixed target sample delivery scheme utilizing the Roadrunner fast-scanning system and ultra-thin polymer/graphene support films, providing a low-volume, low-background approach to membrane protein SFX. This study represents initial steps in obtaining structural information for ApoA1 and ApoE4 NLPs and developing this system as a supporting scaffold for future structural studies of membrane proteins crystalized in a native lipid environment. 

Researchers studying children’s reports of sexual abuse have focused on how questioners overtly assess coaching and truthfulness (e.g., “Did someone tell you what to say?”). Yet attorneys, and defense attorneys, in particular, may be motivated to ask about suggestive influence and truthfulness in subtle ways, such as with implied meaning (e.g., “Did your mom help you remember?”). Such questions may be particularly challenging for children, who may interpret statements literally, misunderstanding the suggested meaning. The purpose of this study was to examine and categorize how attorneys’ ask about suggestive influence and truthfulness. We wanted to learn how attorneys subtly accuse suggestive influence, and how frequently this occurred. We hypothesized that questions indirectly accusing suggestive influence would be common, and that defense attorneys would ask more subtle questions, and fewer overt questions, than prosecutors. We examined 7,103 lines of questioning asked by prosecutors and defense attorneys to 64 children testifying about alleged child sexual abuse. We found that 9% of all attorneys’ lines of questioning asked about suggestive influence or truthfulness. The majority (66%) of these were indirect accusations. Indirect accusations of suggestive influence spanned a range of subtleties and topics, including addressing conversational influences (e.g., coaching), incidental influences (e.g., witnessing abuse), and others. We also found defense attorneys were less likely than prosecutors to ask about suggestive influence and truthfulness overtly. We conclude that attorneys commonly ask about suggestive influence and truthfulness in subtle ways that developing children may struggle to understand, and which may result in affirmations of influence, even when allegations are true.